Device and surgical system for rapid aseptic isolation and concentration of autologous fat derived stem cells

ABSTRACT

The present disclosure provides an apparatus for harvesting stem cells from fat tissue. The apparatus may include a first transducer coupled to a first end of an resonant horn to form an ultrasonic resonator. The resonant horn may include an elongated body having a plurality of through-holes configured to accommodate a plurality of specimen containers that are positioned substantially perpendicular to the elongated body. The apparatus may also include a wave generator coupled to the first transducer to generate an ultrasonic wave, wherein the elongated body has a length that is multiples of the half-wavelength of the ultrasonic wave.

CROSS-REFERENCES TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/016,561 filed Feb. 5, 2016, which application claims priority toU.S. Patent Application No. 62/113,425, entitled “Device and SurgicalSystem for Rapid Aseptic Isolation and Concentration of Autologous FatDerived Stem Cells,” filed on Feb. 7, 2015. Both applications areincorporated herein by reference in their entireties.

FIELD

The present disclosure is directed to devices and methods for extractionof stem cells from a harvested fat sample from a patient and thereintroduction of the extracted stem cells into the patient. The entireprocedure of extraction is sterile, fast, and cost effective.

BACKGROUND

Stem cell research and interest has been accelerating in recent yearssince methods of identification have been developed. Certain stem cellswithin the body have the potential to differentiate under properenvironment into different cell lines. The stem cells, that are able todifferentiate under proper environment into different cell lines, aregenerally dispersed in many tissues and not readily identifiable in vivonor readily concentrated for clinical applications outside sophisticatedcellular laboratories. There has been an increasing interest in medicalapplications and hope of promoting healing of damaged organs andregenerating tissue in areas like damaged hearts, kidney, or evennerves. Nerve tissue damage is often devastating to the individual untilrecently, as the nerve tissue damage has been thought as non-repairable.The stem cells have been found in embryos and umbilical cord blood.However, such sources were challenged by ethical issues and issues ofantigenic compatibility between donor and recipient cell lines. In morerecent years, autologous sources of pleuro-potential cells were found inbone marrow, blood, and fat. For autologous applications, blood, bonemarrow, and fat offer a readily accessible tissue with minimal morbidityin harvesting the stem cells.

When the fat tissue is broken down, the lipid containing component isremoved. The residual is referred to as a stromovascular component thatcontains numerous mesenchymal derived pluripotential cells. Thestromovascular component including concentrated stromaovascular cells,which are of great interest for their potential in tissue regenerationin variety of degenerative clinical conditions such as osteoarthritis,chondromalacia, cardiovascular, and neurological conditions. Numerousstudies also suggest that the regional effect of surrounding tissue inneed of repair plays a role in cellular differentiation as well ascirculating factors described as growth factors.

Existing approaches in autologous concentration of fat derived stemcells, also referred to as a stromovascular layer or component, arebased on enzymatic breakdown of supportive structures and small vesselswere the pleuro-potential cells are thought to reside within fat.Protocols for clinical use harvest stromaovascular cells or stem cellsfrom fat tissue of a patient, and subject the fat tissue to an enzymaticbreakdown, followed by a centrifuge cycle to extract the stromovascularcomponent or stromaovascular cells, and then mixing the stromovascularcomponent with Platelet Rich Plasma (PRP) derived from blood to containa variety of stimulating growth factors. Some approaches also subjectthe mixture of stromovascular component with PRP to lightstimulation/activation of underlying peptide factors before reinsertionof the mixture into the patient. Recent studies in Korea and UCLAidentify a subset of pleuro-potential cells within fat called Muse-ATthat appear to have greater differentiation and repair capability thanother mesenchymal derived cells.

The conventional process not only introduces reagents to the fat sample,but also has challenges in proper processing and retention of sterilityif such stromovascular cells were to be re-injected into the samepatient. Specifically, by using collagenase or similar enzymes tobreakdown the supportive structures in fat tissue or fat sample,followed by centrifugation, the stromovascular cells can be concentratedand used in numerous emerging clinical applications. The aboveextraction for a viable stromovascular component from fat tissue isgenerally a cumbersome laboratory process, which introduces enzymaticcomponents and also includes manual handling steps that challenge thesterility of the stromovascular component that is reintroduced into thepatient, if the intent is to graft the processed stromovascularcomponent back to the patient from whom a fat tissue is collected andthe cells are harvested from the fat tissue.

There is a rapidly expanding interest in developing autologous stem celltreatments for various disorders affecting mankind. However, a rapid,cost effective, safe, and bedside device has yet not been developed.Accordingly, there is a need in developing the device and systemdescribed hereforth.

BRIEF SUMMARY

The present disclosure provides devices and methods that allowclinicians to rapidly extract a stromovascular component orstromovascular cells from a surgically harvested fat sample. Thesestromovascular cells are viable, autologous, and sterile aftercompletion of the extraction process. These laboratory devices can bereadily completed by any support staff in a clinical facility or pointof care without any specialized laboratory training. The device does notinclude any direct specimen handling that may inadvertently impactsterility. In an embodiment, an apparatus is provided for harvestingstem cells from fat tissue. The apparatus may include a first transducercoupled to a first end of an resonant horn to form an ultrasonicresonator. The resonant horn may include an elongated body having aplurality of through-holes configured to accommodate a plurality ofspecimen containers that are positioned substantially perpendicular tothe elongated body. The apparatus may also include a wave generatorcoupled to the first transducer to generate an ultrasonic wave, whereinthe elongated body has a length that is multiples of the half-wavelengthof the ultrasonic wave.

In an embodiment, an apparatus is provided for extracting stem cellsfrom fat tissue. The apparatus may include a specimen container having aclosed bottom and an open top. The apparatus may also include anelectromechanical agitator inside the specimen container. The apparatusmay further include an agitator cap removably coupled and sealed to theelectromechanical agitator and configured to seal the top of thespecimen container to maintain specimen sterility. The apparatus mayalso include an aspiration cap removably coupled and sealed to thespecimen container to maintain specimen sterility, the aspiration capincluding a syringe with a fluid channel for specimen extraction fromthe specimen container.

In an embodiment, a method is provided for sterilely extracting stemcells from fat. The method may include placing a specimen containercontaining fat in an ultrasonic resonator and applying an externalultrasonic field to the fat to separate stem cells from the fat in amedical care room. The method may also include placing the specimencontainer in a centrifuge to concentrate the separated stem cells andremoving the specimen container from the centrifuge. The method mayfurther include extracting the concentrated stem cells from the specimencontainer. The specimen retains sterility through the sequence of theabove steps.

In an embodiment, a method is provided for extracting stem cells fromfat. The method may include placing an agitator cap onto an open end ofa specimen container having an open end and closed bottom. The agitatorcap may be removably coupled to the specimen container and also coupledto a mechanical agitator. The method may also include separating stemcells from fat in a specimen container by the mechanical agitator in anoperating sterile room. The method may further include replacing theagitator cap with an aspiration cap onto the open end of the specimencontainer. The aspiration cap may be removably coupled to the specimencontainer and also coupled to a syringe configured for extractingsamples from the specimen container. The method may also include placingthe specimen container with the aspiration cap in a centrifuge toconcentrate the separated stem cells, removing the specimen containerfrom the centrifuge, and followed by extracting the stem cells from thebottom of the specimen container. The specimen retains sterility throughthe sequence of the above steps.

In an embodiment, a method is provided for extracting stem cells fromfat. The method may include getting a fat sample from a patient using asyringe with vacuum assistance and electromechanically agitating the fatsample in a tube with a sealed cap in a medical care room. The methodmay also include centrifuging the agitated fat sample in a sealed tubeto retain sterility. The method may further include extracting thestructural fraction containing stem cells from the tube in the medicalcare room, and reintroducing the stem cells to the same patient in themedical care room.

In an embodiment, a method is provided for extracting stem cells fromfat tissue. The method may include agitating the fat tissue in a sealedcontainer to produce a stromovascular component containing stem cells.The method may also include centrifuging the sealed container toconcentrate the stromovascular component. The method may further includeextracting the concentrated stromovascular component from the sealedcontainer. The fat tissue and the stromovascular component remainsterility in the sealed container.

Additional embodiments and features are set forth in part in thedescription that follows, and will become apparent to those skilled inthe art upon examination of the specification or may be learned by thepractice of the disclosed subject matter. A further understanding of thenature and advantages of the present disclosure may be realized byreference to the remaining portions of the specification and thedrawings, which forms a part of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The description will be more fully understood with reference to thefollowing figures and data graphs, which are presented as variousembodiments of the disclosure and should not be construed as a completerecitation of the scope of the disclosure, wherein:

FIG. 1 shows an external ultrasonic agitator system in accordance withembodiments of the present disclosure.

FIG. 2 shows a prototype of a resonant horn in accordance withembodiments of the present disclosure.

FIG. 3 shows a countertop ultrasonic agitator device that includes thehorn of FIG. 2.

FIG. 4A is a side sectional view of a specimen container including aplurality of ridges on an inner wall in accordance with embodiments ofthe present disclosure.

FIG. 4B is a top view of the specimen container of FIG. 4A.

FIG. 4C is a top view of an alternative design of the specimen containerincluding grooves and ridges in accordance with embodiments of thepresent disclosure.

FIG. 5 illustrates an example electromechanical rotatory agitator deviceincluding a tissue container and an agitator cap coupled to an agitatorprobe in accordance with embodiments of the present disclosure.

FIG. 6 illustrates the tissue container of FIG. 5 and an aspiration capcoupled to a syringe drawing port to replace the agitator cap of FIG. 5in accordance with embodiments of the present disclosure.

FIG. 7 illustrates a conventional tube with an agitator cap inaccordance with embodiments of the present disclosure.

FIG. 8 is a flow chart that illustrates the steps of a medical procedurein accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION

The present disclosure may be understood by reference to the followingdetailed description, taken in conjunction with the drawings asdescribed below. It is noted that, for purposes of illustrative clarity,certain elements in various drawings may not be drawn to scale.

Adipose tissue is an abundant, accessible, and replenishable source ofadult stem cells that can be isolated from liposuction aspirate andwaste tissue. A conventional method of isolating stem cells relies on acomplicated ex vivo treatment with collagenase and exogenous digestiveenzymes.

The present disclosure provides electromechanical devices and methodsfor harvesting the pluripotential cells from fat tissue, which is areadily available adult tissue source. The electromechanical devicedescribed herein includes an electromechanical agitator designed todisrupt and homogenize harvested fat tissue and to extract thestromovascular component from the fat tissue. The present disclosurealso provides a centrifuge for concentrating the separated stem cells.The centrifuge is operated in a temporal sequence with theelectromechanical device.

In some embodiments, the electromechanical devices may include anexternal ultrasonic agitator that includes a resonant horn toaccommodate specimen containers. The present disclosure also provides aspecimen tube that includes ridges or grooves for acoustic impedancematching between the horn and the fat tissue.

The ultrasonic energy in the external ultrasonic agitator system createsultrasonic cavitation in the fat tissue, which accomplishes dispersing,de-agglomerating, disrupting and disintegrating of adipose cells priorto the centrifugal step. The external ultrasonic agitator systemutilizes intense ultrasonic cavitation and mechanical forces to separatestem cells and stromal vascular fraction from white adipose tissue orfat cells. Since strong cavitation may lyse fat cells and red bloodcells, the resulting hematopoietic stem cells, plasma platelets andother health factors of lipo-aspirate are retained in the final product.The ultrasonic procedure can avoid unnecessary laboratory and chemicalor biological manipulations. The ultrasonic procedure is simple,straightforward, and sterile and provides isolation of stem cells fromfat produced in accordance with FDA 361 and Good Manufacture Practiceguidelines. This ultrasonic procedure is very fast and thus allowsextracted stem cells to be used off-label and at the discretion ofattending physicians. Since the mesenchymal stem cells and stromalvascular fraction are extracted from the patient, it is also autologouswith minimal risk of an immune response, showing potential for directedclinical therapy against a variety of disease and damaged tissue.

The required energy density can be achieved through integration of thespecimen vials or tubes directly into the mechanically fine-tunedultrasonic horn. For a high intensity ultrasound generator, a resonanthorn is an element that transfers mechanical energy from the oscillatingpiezoelectric transducers to a work piece, such as specimen tubes orvials. For example, each of two or four polyvinyl caped vials maycontain adipose tissue fluid removed from abdominal lipo-suctionprocedure. Liposuction source may include other parts of the bodybesides the abdomen.

In some embodiments, the electromechanical devices may include anagitator probe that can contact the fat tissue inside the specimencontainer. The agitator probe may cause rotatory or vibrationaryagitation inside the specimen container. The devices may include anagitator cap removably coupled to seal the specimen container foragitating the sample. The devices may also include an aspiration capwhich can replace the agitator cap for centrifuging the agitated sample.The present disclosure also provides a specimen tube that includesridges or grooves for improving efficiency of the agitation.

The present disclosure also provides a sterile method for harvesting fattissue from a patient and extracting stem cells from the fat tissue andreintroducing into the patient in 15-30 minutes. The sterile method usesan intense ultrasonic excitation, or rotatory or vibratory agitation,followed by a differential centrifugation, which is a simpler, moreeffective, minimally invasive and sterile procedure for more efficientrecovery of adipose somatic stem cells and stromal vascular fractionthan conventional techniques.

The following section describes the design features and functionality ofan external ultrasonic agitator device that can adjust ultrasonicfrequency and amplitude for optimal settings useful for clinicalapplication as described herein. The electromechanical device describedherein combined with the described process can greatly simplify theavailability of mesenchymal stem cells derived from fat in real time theclinical setting. Preservation of sterility, minimizing use of externalagents, and instrumentation that can be used by any hospital personnelare important practical features. The extraction process of stem cellsfrom a fat sample may be completed in about 15-30 minutes. The presentdisclosure provides clinical investigators with an effective tool forrapidly extracting pluripotential cell pellet from harvested fat samplethat is often discarded. The device can greatly improve the efficiencyof extraction and is user friendly for such applications. A method forcellular anatomical localization of the derived product is described.

An external ultrasonic field may be applied to a sealed fat containingtube. The electromechanical vibratory agitator provides ultrasonic poweramplification by resonance. The electromechanical vibratory agitatorincludes a horn for amplifying the ultrasonic power. The agitatorprovides amplified ultrasonic power that is used for the break-down offat samples.

For a conventional ultrasonic activation of vials in an ultrasoniccleaning tank, the water surrounding the vials takes most of the energy.The power in a 50 mL vial is less than 0.1 watt for ultrasonic cleaning.For ultrasonic cell separation processes, such a low power level isineffective. Thus, a power boost for ultrasonic energy is used for stemcell isolation and recovery. The power boost may exceed 500 times.

FIG. 1 shows an external ultrasonic agitator system in accordance withembodiments of the present disclosure. External ultrasonic agitatorsystem 100 includes two transducers 106A-B firmly attached to a resonanthorn 104 that may be light and rigid. Each of transducers 106A-B may beformed of multilayer piezoelectric crystals 112. The resonant horn 104can accommodate aseptic tissue containers 102 and can also amplify andtransfer ultrasonic energy from the transducers 106A-B to the aseptictissue containers 102. The horn 104 may include dual opposing ends114A-114B. Each of ends 114A-114B is connected to one side of therespective transducer 106A or 106B. The horn 104 has a length ofmultiples of ½ wavelength.

The horn 104 includes an elongated body extending along a longitudinalaxis 116 between two opposed ends. The horn includes elongated holes 118that are transverse to the longitudinal axis. The resonant horn 104 canaccommodate four tissue containers or specimen tubes 102 in thisexample. In other embodiments, the horn may include more or fewer holes118 to accommodate more or fewer tubes 102. The resonant horn 104 may beformed of a light weight metal, such as titanium or aluminum.

The external ultrasonic agitator system 100 may also include an outerbacking end 110A-B on each outer side of the respective transducers106A-B. Each of the outer backing ends 110A-B is configured to transmitthe ultrasonic vibration only toward the horn 104.

The external ultrasonic agitator system 100 may also include a powergenerator module or wave generator 108 that drives the piezoelectrictransducers 106A-B. For example, the power generator module 108 may be aphase shifted square wave generator which is electrically coupled to thepiezoelectric drivers. The phase shifted wave generator 108 can becontrolled to generate two phase-shifted waves. The wave generator 108may produce a standing wave in the horn 104. The wavelength may be about20 cm at 25 kHz in the horn 104 that has a sound velocity of about 5000m/s.

The wave generator 108 can also be self-tuned to the tissue tube orcontainer including specimen. In a particular embodiment, the externalultrasonic agitator system 100 may include a self-tuning circuit that isbased on a power current plateau. The system 100 may also include aself-tuning circuit with an acceleration sensor to boost optimalresonance.

The tissue containers or specimen tubes 102 are positioned at thelocations in the horn for maximum ultrasonic power. Generally, thenumber of tubes in the resonant horn may be an even number, for example,at least 2, 4, 6, among others, such that the centrifuge head iscounterbalanced with the even number of tubes that have filled specimen,such as fat tissue.

The frequency in the system 100 may be tuned to break apart the largercollagen stroma and yet preserve the viability of the smallerstromovascular cells.

The vibratory energy or amplitude in the system 100 may also bemodulated by a heat measuring servo mechanism, which is implemented intothe power generator module or wave generator. The system 100 may includea temperature sensor (not shown) for measuring the temperature of thehorn 104 near the specimen tubes 102. The sensor can measure specimentemperature in the specimen container during ultrasonic disruption. Thetemperature sensor can provide feedback for the system 100 to adjust theoutput of the wave generator 108 to prevent the tissue specimen fromoverheating. For example, if the temperature exceeds 43.5° C., thevibratory amplitude will be reduced, because the excess ultrasoundenergy may damage the viability of the cells.

The system 100 may include a self-tuning capability and subject thesealed specimen tubes to high fluxes of ultrasonic dispersion/separationenergies. The system 100 may include an acceleration sensor coupled tothe horn to provide feedback for self-tuning and optimum power output.

In an alternative embodiment, one transducer may be attached to an endof the horn to form a standing wave in the horn. One opposing end of thehorn is configured to reflect the ultrasonic wave generated by thetransducer. FIG. 2 shows a prototype of a resonant horn in accordancewith embodiments of the present disclosure. As show, one transducer 206including four piezoelectric crystals 212 is attached to only one end ofthe horn 204. Also, the horn 204 accommodate two specimen tubes (notshown). The ultrasonic wave generated by the transducer 206 is reflectedfrom an opposing end with a phase difference of about 180° C. from theincoming wave generated from the transducer 206. The reflected wave andthe wave generated from the transducer 206 forms a standing wave.

The horn 204 may include a first portion 204A attached to one of thecrystal disks 212 to the left side of the horn 204. The first portion204A has a diameter close to the crystal disks 212 that transmitultrasonic waves along a longitudinal axis 116. The horn 204 alsoincludes a second portion 204C which has a smaller diameter than thefirst portion. The horn 204 also includes a transition portion 204Bbetween the first portion 204A and the second portion 204C. Thetransition portion 204B has a larger diameter than that of the firstportion 204A and is smoothly transitioned from the second portion 204C.The horn 204 also includes a third portion 204D coupled to the secondportion 204C. The third portion 204D has a larger diameter than thecrystal discs 212 and includes two through-holes 218A-B that are spacedapart along the longitudinal axis 116 and accommodate two specimentubes, respectively.

It will be appreciated by those skilled in the art that the number ofcrystal disks in the transducer may vary. The higher number of crystaldisks may generate high ultrasonic energy. The crystal disk thicknessmay vary to provide desired ultrasonic frequency.

The piezoelectric transducer 206 may be firmly attached to one end ofthe horn and may be driven by a square wave power generator module 108.The ultrasonic wave may travel in the horn at a velocity of 5000m/second and with a wavelength of 20 cm for a frequency of 25 kHz. Inthis example, the horn 204 can accommodate two specimen tubes. Thelength of the horn is a half-wavelength multiplier. The phase shiftedwave generator 108 can be adjusted to form an ultrasonic resonator inthe horn 204.

The ultrasonic transducer 206 may include PZT5 piezoelectric crystaldiscs that are clamped together by a strong bolt to form a serialmechanical oscillator, which has vibration amplitudes from each crystaldisc reinforced in synchronization. The crystal discs 212 areelectrically connected in parallel and are driven by high voltage pulsewave signals. The driving voltage wave signals may be maintained at amechanical resonance frequency of the piezoelectric crystals 212.

FIG. 3 shows a countertop ultrasonic agitator device 300 that includesthe horn 204 of FIG. 2. The countertop ultrasonic agitator device 300has an upper portion 302 that includes an ultrasonic transducer 206consisting of piezoelectric crystal disks 212. One of the crystal discs212 is connected to a resonant horn 204, which amplifies the mechanicaldisplacement of the ultrasonic oscillation from the crystal discs 212.

The horn 204 may be constructed of a strong and lightweight metal alloy,such as titanium alloy or aluminum alloy, to transfer mechanicalvibration from transducers to the specimen tube. The length of the horn204 between two opposing ends 214A-B may be configured in relation tothe wavelength of the traveling ultrasonic wave. For example, the lengthmay be a multiple of the half wavelength traveling in the horn 204.

The countertop ultrasonic agitator device 300 also has a lower portion304 that includes the resonant horn 204 to accommodate two specimentubes 102. At the bottom end 214B of the horn 204, the countertopultrasonic agitator device 300 includes a backing 210B that reflects theultrasonic wave generated by the transducer 206. The backing 210B isconfigured to provide the wave reflection in roughly 180° for theultrasonic wave generated from the transducer 206 above a top end 214Aof the horn 204 and transmitted through the horn 204 toward the backing210B at the bottom of the device 300. The reflected wave from thebacking 210B forms a standing ultrasonic wave with the ultrasonic wavegenerated by the transducer 206. Another backing 210A attached to thetransducer 206 is configured to have the transducer vibrate only towardthe horn.

The countertop ultrasonic agitator device 300 may also include aseparation plate 306 that separates the upper portion 302 from the lowerportion 304. The transition portion of the horn 204 sits on top of theseparation plate 306.

Two specimen vials are placed in locations where the local mechanicalvibrations and cavitation action are maximized. The oscillationfrequency may vary with the dimension and material of the piezoelectrictransducer. The amplitude of the ultrasonic transducer can be adjustedby varying the power of the ultrasonic generator in order to vary themechanical amplitude of vibration or ultrasonic energy into the specimentube.

Specimen Tube

The tissue container 102 may be a specimen tube with inner wallfins/grooves for improved ultrasonic coupling of the fat tissue withexternal energy sources. The shape and walls of the tissue container canbe optimized for maximum transfer of externally applied ultrasonicdispersive energy. For example, the container may include internal wallprojections into the specimen containing lumen for impedance matchingbetween the horn and the fat tissue.

FIG. 4A is a side sectional view of a specimen container including aplurality of ridges on the inner wall in accordance with embodiments ofthe present disclosure. FIG. 4B is a top view of the specimen containerof FIG. 4A. FIG. 4C is a top view of an alternative design of thespecimen container including grooves and ridges in accordance withembodiments of the present disclosure.

As shown in FIGS. 4A-4C, the internal ridges or fins 402 are radiallyinwardly projected from the wall 406 of the tube 102 and are orientedalong the longitudinal axis 412 of the tube 102. The outer grooves 404open radially outwardly from the outer wall 408 of the tube 102 and areoriented along the longitudinal axis 408 of the tube 102. These ridges402 or grooves 404 help provide acoustic coupling between the horn andthe fat tissue inside the lumen 410 of the tube 102 for optimalultrasonic energy transfer.

The geometry of the ridges, fins, and/or grooves may be tuned foroptimal energy coupling between the horn and the semi-liquid contentsinside the tube. In a particular embodiment, the specimen tube 102 mayinclude a specially designed conical end, like the ones used in largercentrifuges. It will be appreciated by those skilled in the art that theshape of the specimen tube may vary. The configuration of fins orgrooves of the specimen tube may also vary, such as shapes of the finsor grooves, spacing between the fins or grooves or arrangement of thefins or grooves.

These tubes 102 are also configured to have minimal sound reflectionwhich may lead to destructive cavitation effects and cellular damage tothe stromovascular component.

These specimen tubes 102 may be constructed from plastic, such aspolyethylene, or any material that is not brittle, but is sufficientlyrigid, and is also semitransparent for the fat sample in the tube to bevisible. These specially designed specimen tubes 102 are sterile and maybe supplied to the surgeon for harvesting fat tissue in the medical careroom or unit, such as an operating room, a medical doctor office, or anemergency room among others. The specimen tubes with ridges and/orgrooves 102 may be injection molded.

As an example, a sample vial may be a 50 mL conical centrifuge tube withan aspiration cap of 30 mm diameter. The vial may be about 115 mm longand have a conical bottom tip. The sample vial is autoclavable andfreezable.

Electromechanical Agitator Device with Agitator Cap and Aspiration Cap

In an alternative embodiment, the electromechanical agitator device mayinclude a rotatory agitator probe, or a vibratory or linearpiezoelectric specimen agitator to provide sufficient energy to createcavitation in tissue in the tube. The rotatory agitator probe may beinserted into the tube and may be in direct contact with the fat.

The tube may be a conventional tube without any ridges, fins, orgrooves. Alternatively, the tube may be specially designed to includeridges, fins and/or grooves for improved efficiency, as disclosed inrelated texts with respect to FIGS. 4A-4C. The tube may also be modifiedby including a number of metal rods that act like the ridges or fins, asdisclosed in detail with respect to FIG. 7 later.

In some embodiments, the rotatory agitator probe may be driven by anultrasonic or subsonic rotary electromechanical engine (e.g. electricmotor) for breakdown of collagen/fibrous structure to accomplish tissuedispersion to separate stromovascular cells. In alternative embodiment,the linear piezoelectric specimen agitator may include a vibratoryelectromechanical engine for the breakdown of collagen/fibrousstructure. The agitator probe may include rotary or oscillating enginesthat are used for surgical bone drilling or cutting instrumentation.Examples of such readily available electromechanical engines are theSTRYKER@ microdrill, STRYKER@ impaction drill, Medtronic Xomed XPS powerset, Medtronic Triton system and variety of similar powerelectromechanical engines readily available in most existing operatingroom suites.

FIG. 5 illustrates an example electromechanical rotatory agitator deviceincluding a tissue container and an agitator cap coupled to an agitatorprobe in accordance with embodiments of the present disclosure. Theelectromechanical rotatory agitator device 500 includes a tissuecontainer or specimen tube 102 removably coupled to a sealed agitatorcap 502. The tube 102 includes an open end 514. The agitation cap 502 issealed to the open end of the tube. With the agitator cap, the specimentube can be sealed for tissue agitation, tissue dispersion, tissueemulsion, or tissue shearing.

The electromechanical rotatory agitator device 500 also includes anagitator probe having a number of rotors 510 attached to a rotary shaft508, which is coupled to the agitator cap 502. The agitator cap 502 mayinclude a central hole configured to allow the rotatory shaft 508 toextend inside the lumen 410 of the tube 102.

The electromechanical rotatory agitator device 500 also includes asealed sleeve bearing 506 coupled between the rotary shaft 508 and theagitator cap 502 to allow the rotary shaft to freely rotate and also toseal the central hole of the agitator cap 502. The sleeve bearing 506may be in a tube form or ring form. The sleeve bearing 506 may have aninner diameter larger than the outer diameter of the rotary shaft 508.The sealed sleeve bearing 506 may be formed of a plastic with lowfriction, such as polytetrafluoroethylene (PTFE). The sealed bearing 506is embedded in the agitator cap 502.

On two opposite sides of the agitator cap 502, one stopper 504B iscoupled to the rotatory shaft 508 inside the lumen 410 of the tube 102and another stopper 504A is coupled to the rotary shaft 508 outside thetube 102. The two stoppers 504A-B are configured to prevent the rotatoryshaft 508 from movement along the longitudinal axis 412. The rotatoryshaft 508 can be removed such that the sleeve bearings 506 can bethoroughly cleaned.

In some embodiment, a stabilizer may be added at the end of the rotatoryshaft outside the agitator cap to further stabilize the rotation of therotor if necessary.

The ridges or fins on the inner wall (as shown in FIG. 5) or the grooveson the outer wall of the tube (similar to that shown in FIG. 4C) mayprovide improved efficiency of rotary blender function. The geometry ofthe ridges, fins and/or grooves may be tuned for optimal energy couplingbetween the horn and the semi-liquid contents inside the tubes. In someembodiments, fins may be used inside the tube. In some embodiments,grooves may be used outside the tube. In some embodiments, a combinationof fins and grooves may be used in the tube.

In a particular embodiment, the specimen tubes may include a speciallydesigned conical end 512, like the ones used in larger centrifuges. Itwill be appreciated by those skilled in the art that the shape orconfiguration of the specimen tube may vary.

FIG. 6 illustrates the tissue container of FIG. 5 and an aspiration capcoupled to a syringe drawing port to replace the agitator cap of FIG. 5in accordance with embodiments of the present disclosure. The tube 102with the aspiration cap 602 can be placed in a centrifuge system tocomplete the separation phase. The aspiration cap 602 is coupled to asyringe drawing port 606 connected to a center cannula 608, which isused to aseptically aspirate the concentrated stromovascular cellularcontent at the bottom 512 of the tube 102 at the completion of thecentrifuge stage described herein.

The aspiration cap 602 is also coupled to an air inlet 604, which allowsair to flow inside the tube. The air inlet 604 keeps the lumen 410 ofthe tube 102 at an atmospheric pressure after the extraction of the stemcells from the bottom 512 of the tube 102 through the syringe drawingport 602 and the center cannula 608, such that specimen extraction canbe performed without generating undue negative pressure within thespecimen container.

The specimen tube 102 of FIGS. 4A-4C, with the aspiration cap 602 asshown in FIG. 6, can be used in the system 100. Throughout theultrasonic dispersion process in system 100, the tube 100 is sealed withthe aspiration cap 602 such that the fat tissue inside the specimen tuberemains sterile.

FIG. 7 illustrates an electromechanical rotary agitator device includinga conventional tube with an agitator cap in accordance with anembodiment of the present disclosure. The conventional tube 706 does notinclude any ridges or fins as shown in FIGS. 4A-C, FIG. 5 or FIG. 6. Asshown in FIG. 7, the agitator device 700 includes a specimen tube havinga closed bottom and an open end sealed with an agitator cap 702. Theagitator device 700 also includes rotors 710 coupled to a stator 708that is driven by an electric motor (not shown). The rotors 710 agitatethe fat tissue inside the tube 706.

The agitator cap 702 includes a central through-hole through which astator rod 708 extends into the specimen tube. The stator rod 708includes an outer end extending outside the agitator cap 702. The statorrod 708 also includes an inner end that extends through the centralthrough-hole of the agitator cap 702 toward the bottom 712 of the tube706, but does not contact the bottom of the tube. The outer end of thestator rod can be coupled to an electric motor to cause the rotation ofthe rotors.

Inside the tube, a plurality of rotors 710 are coupled to the stator rod708 and are spaced apart from each other. The rotors 710 are driven torotate by the electric motor coupled to the outer end of the stator rod708. It will be appreciated by those skilled in the art that the rotorsmay vary in shapes and the number of rotors to accomplish optimalbreak-down of the fat tissue in the tube.

In the conventional tube, a few stabilizing metal rods or bars 714 maybe added to act like the ridges or fins inside the tube to help improvethe efficiency of the mechanical agitation of the fat tissue. The metalrods 714 have one end coupled to the agitator cap 702 and one endextending toward the bottom 712 of the tube 706, but does not contactthe bottom. The stabilizing bars also provide counter spin stabilizationof specimen within the specimen container.

Because of the sample in the sealed tube 706 or container remainssterile, the aspiration of the concentrated stromovascular cellularcontent is performed by using a syringe and a lock system to maintaincontent sterility. The tube 706 includes the agitator cap 702 coupled tothe stator rod 708 to which the rotors 710 are attached. Thus, thismethod does not require introduction of any new molecules into theprocessing of fat tissue and eliminates the possibility of viral orbacterial contamination of the specimen. The specimen reintroduced tothe patient is genetically autologous, contains only the original tissuecomponents, and thus obviates possibility of transmissible organisms.

The agitation for tissue dispersion and isolation can be performed by ahealth provider in existing and certified operating room facilities,such as an operating room, an emergency room, or a medical office or amedical care room or unit. After the agitation, the tube is handed offthe field and given to the support staff for the centrifuge.

Example Ultrasonic Resonant Agitator System

An example ultrasonic horn may be about 380 mm long, 108 mm wide, and165 mm in height with a side loading. The example resonant horn may holdtwo 50 mL caped vials.

An example wave generator or power unit may be about 280 mm long, 203 mmwide, and 159 mm in height. The wave generator uses AC power, forexample, 110 V AC×5 A×60 Hz (Circuit breaker rating). The transducer mayhave a frequency of 25.09 KHz+/−50 Hz.

The transducer may include four crystal discs that may be formed of PZT5piezoelectric crystals. Each disc may have a diameter of 50 mm and athickness of 6.5 mm. The four crystal discs may be pre-stressed via acenter compression bolt. The ultrasonic resonant agitator system mayhave a maximum capacity of 500 W.

The power unit may include a power on/off switch and has a SonicActivation Duty Cycle ranging from 10% to 100% (10% to continuous). Thepower unit may also include a Sonic Activation ON/OFF switch. The sonicenergy can be adjusted by a dial setting. The electrical wave amplitude(sonic energy) can be adjusted. The power unit may also include aFrequency Fine Tuning.

The ultrasonic resonant agitator system may adjust its temperature. Theultrasonic resonant agitator system may include a safety circuit inreal-time by using a temperature sensor (e.g. an IR sensor orthermocouple) for temperature measurement that provides feedback to thesystem to control the duty cycle of the wave generator or power unit.The temperature sensors, such as thermocouples, may use an isolatedbattery power source.

The ultrasonic resonant agitator system may also include a display. Forexample, the power unit may include a front panel that displays a greenlight indicating AC Power “ON” light. The power unit may also include aSonic Activation Status indicator light yellow and indicate Ultrasound“ON.” The power unit may also include a red light for indicating aspecimen tube or vial being overheated. The power unit may also includerear panel input/output connections and an exhaust fan.

The ultrasonic resonant agitator system may include a display of vialtemperatures. For example, an LCD digital display may show temperaturesof vials or a temperature limit setting at an accuracy of 0.1° C.

The operating procedure may include the following steps: (1) keep thepower unit power on/off switch in an “OFF” position; (2) connectinterface and sonic power cables between the power unit and ultrasonichorn; (3) preset the power unit controls including turning Sonic Energy& DC Bias Adjust to minimum, turning ultrasound toggle switch to an“OFF” position, selecting desired duty cycle, setting Frequency FineTuning knob to a mid-position, and inserting two sample vials containingliquid specimen.

For testing, the power unit is turned to an “ON” position to observe thefollowing normal displays and indications: green power-on light is on;ultrasound status light is off; and the ultrasonic horn displays correctvial temperature readings after a brief thermocouple sensor start-upperiod to have up to 2° C. accuracy.

The next step is to adjust the desired temperature limit for theresonant horn by depressing the toggle switch and turning the limitadjustment knob simultaneously and to release the toggle switch and lockadjustment knob after the desired limit is obtained. Note that when thevial temperature exceeds the limit, sonic energy is momentarilyinterrupted until vial temperature drops below set limit, such as 2° C.below. The Sonic Activation Status indicator light on power unit willturn red during this period.

The operating procedure may include:

(1) Turn Sonic Energy & DC Bias Adjust knob clockwise for a meterreading of approximately 250 VDC (dial setting of 5);

(2) Turn ultrasound ON/OFF toggle to “ON” position. The sonic energy andsonic Activation Status indicator light (yellow) turns on within 10seconds;

(3) During the period when sonic energy and yellow light is on, adjustthe Frequency Fine Tuning knob (clockwise initially) for a maximum dip(approximately 50V) in DC Bias Voltage, indicating resonant oscillationof the horn. For out-of-tune frequencies, there is little or no dip inDC Bias Voltage, indicating little or no horn oscillations. Furtherfine-tuning can be obtained by observing the frequency of strayelectrical signals picked up by a nearby oscilloscope. Note that theliquid specimen property and filling level in vial may affect thefrequency setting slightly;

(4) Reduce or re-adjust Sonic Energy & DC Bias Adjust knob for thedesired sonic energy;

(5) Re-select the desired duty cycle other than the 1.0 “continuous”initially selected. The duty cycle is based on a 10-seconds ON/OFF clockperiod. The new duty cycle selected may take effect in a maximum delayof 10-seconds;

(6) Note that there is a built-in overheat safeguard for internal powerdriver devices. Sonic energy is interrupted until overheating conditionis off. Such overheating occurs when the DC Bias Voltage setting is toohigh, electrical waveform becomes erratic, and the piezoelectriccrystals cease to function properly;

(7) An isolation transformer inside the Power Unit amplifies the ACpower delivered to the power stage. The 25 KHz square-wave high voltageelectrical signals are delivered to the piezoelectric crystals, whichare electrically isolated from the titanium horn. Note that for furthersafety precaution, skin contact with the horn during operation should beavoided; and

(8) Adjusting the Frequency Fine Tuning knob may encounter thefirst-harmonic resonance oscillation of the device (around 28 KHz).Oscillation intensity would be much lower at higher frequency. TheFrequency Fine Tuning may adjust frequency from 22.4 kHz to 29 kHz in asmall increment by adjusting dial count, as shown in Table 1.

DIAL COUNT Frequency (kHz) 000 29 100 28 200 27.3 300 26.2 400 25.6 50025 600 24.5 700 24 800 23.2 900 22.7 999 22.4Ultrasonic Agitation Process and Applications of Extracted Stem Cellsfrom Fat Tissue

The present disclosure provides devices and methods that can helpclinicians rapidly extract stromovascular cells from harvested fattissue of a patient, process the fat tissue rapidly, and preserve thesterility of the extracted stromovascular cells. The procedure does notrequire any particular laboratory skills, and the procedure does notintroduce foreign substances which could pose safety issues whenreintroduced back into the patient.

Also, sterility is preserved throughout all the steps. Additionally, theprocessing of the fat sample can be performed by medical personnelwithout any laboratory or extensive device training. For these and otherreasons the present device and system possesses compelling advantagesover existing or conventional technologies for concentration ofstromaovascular cellular layer. The present device is better, faster,and cheaper than the conventional technologies for cellularconcentration.

The present disclosure provides an aseptic technique of concentratingstromovascular cells within human fat specimen and a system forautologous re-implantation into the same patient within a period of15-30 minutes without the specimen leaving the operating room site.

The ultrasonic agitation process or dispersion process by using theexternal ultrasonic agitator system 100 or system 300 may take about aperiod of 5-10 minutes at energy levels that may be optimized. After theultrasonic dispersion process, the tube is moved from theelectromechanical agitator to an adjacent centrifuge and is subjected togravitational separation for a period of about 4 to 8 minutes. Aftercompletion of both the vibratory cycle and the centrifugation cycle, thedesired stem cells are aseptically aspirated. A sterile syringe may beused to aspirate the desired contents from the apex of the tube. Thedesired cellular contents can be drawn into the sterile syringe and canbe used by a clinical personnel. The cellular contents or stromovascularcells contain an enhanced number of pleuro-potential cells derived fromthe processed fatty tissue. Because the fat tissue has been mechanicallystressed by the ultrasonic agitation, many of the larger cells mayrelease various growth factors and chemo tactic peptides that furtherenhance the responsiveness of the viable pleuro-potential cells that areconcentrated and reintroduced into the same patient in any desiredanatomical location.

A health provider can therefore readily and aseptically obtain 3 to 5 mLof concentrated stromovascular cells harvested from a sealed tissue tubeor container of approximate 50 mL. About 2 to 3 mL of desired cellularcontents may be produced. For an ultrasonic resonant agitator systemthat can accommodate 2 or 4 tubes, a single processing cycle may yield 5to 10 mL of desired cellular contents or stromovascular cells.

Similarly, the agitation process by the agitator probe with an agitationcap as shown in FIG. 5, followed by the centrifuging step by the probewith the aspiration cap as shown in FIG. 6, may take about less than 30minutes, preferably 15 minutes or 10 minutes. The ultrasonic dispersionprocess or agitation process eliminates the need for introducing reagent(e.g. collagenase or other enzymatic digestion) into the livingspecimen, such as fat tissue.

FIG. 8 is a flow chart that illustrates the steps of a medical procedurein accordance with embodiments of the present disclosure. Medicalprocedure 800 may include getting a fat sample from a patient using asyringe with vacuum assistance at step 802. Medical procedure 800 mayalso include electromechanically agitating the fat sample in a tube witha sealed cap in a medical care unit at step 806. An electric powersource may be provided for agitating the fat sample. This agitating stepmay be accomplished by either the external ultrasonic resonant agitator100 or 300 using the specimen tube as shown in FIGS. 4A-4C. Theagitating may also use the rotatory agitator probe device as shown inFIGS. 5-7, or a linear vibratory probe (not shown). Medical procedure800 may further include centrifuging the agitated fat sample in thesealed container to protect the sterility of the fat sample at step 810,followed by extracting the stromovascular fraction containing stem cellsfrom the tube in the medical care room to protect sterility of thesample at step 810. The sealed container may be the same sealedcontainer for the agitating step, centrifuging step and the extractingstep, thus increasing the likelihood of maintain the sterility. Themedical procedure may also include reintroducing the stem cells to thesame patient at step 818.

The process enables anatomical localization of the cellular graft, asopposed to current indiscriminate intravenous injection of cellularcontent with the hope that the stem cells will migrate to the desiredsite of clinical issue on their own and hypothetical guiding factors.The use of a cellular biological matrix with growth and chemotacticpeptides is sterilely preserved.

In alternative embodiments, the present disclosure provides a biologicalretaining packet with cellular hydration port, which allows the use ofthe extracted stem cells for patients at a later time. Specifically, aregeneration matrix may be packaged in a specially designed pocket witha syringe connector port. The concentrated cellular contents obtainedfrom the ultrasonic dispersion process and the centrifugation cycle canbe injected into the specially designed pocket containing the Biodesigngraft. The injection serves a dual purpose of hydrating the graftmaterial and saturating the graft with enhanced cellular contents. Thesaturated graft can be used to repair tissue in any surgically desiredarea. The regeneration matrix with cellular contents offers enhancedtissue repair at specific anatomical area by virtue that the cellularcontent is anatomically localized and retained in the area of matrixinsertion. In a particular embodiment, a sealed biological retainingpacket containing acellular biological matrix with a cellular hydrationport through which the harvested stem cells within the processedstromovascular fraction containing stem cells can be introduced foradhesion/hydration to the matrix for subsequent replantation.

The product is composed of high viability, mesenchymal stem cells andother important cell populations, including pre-adipocytes, endothelialcells, smooth muscle cells, fibroblasts, immune cells and a variety ofgrowth factors. The product is more therapeutically efficacious thanstem cells alone. It can be used for clinical regenerative healing in avariety of tissues in areas of cosmetic, periodontal medicine,orthopedics, osteoarthritis and other even more important treatments.

The electromechanical and ultrasonic resonator device can be used asalone or can also be used in combination with established enzymaticdigestive process. The electromechanical and ultrasonic technologiesdescribed herein can function as process accelerators. A small amount ofcollagenase mixed with the specimen may accelerate the enzymaticbreakdown of fat or other tissue when subjected to electromechanicalenergies by virtue of micro dispersion as well as direct energyinduction of enzymatic digestion of collagen retaining matrix.Therefore, the present technology can be used in combination with theconventional technology to make the overall extraction significantlymore efficient.

Having described several embodiments, it will be recognized by thoseskilled in the art that various modifications, alternativeconstructions, and equivalents may be used without departing from thespirit of the invention. Additionally, a number of well-known processesand elements have not been described in order to avoid unnecessarilyobscuring the present invention. Accordingly, the above descriptionshould not be taken as limiting the scope of the invention.

Those skilled in the art will appreciate that the presently disclosedembodiments teach by way of example and not by limitation. Therefore,the matter contained in the above description or shown in theaccompanying drawings should be interpreted as illustrative and not in alimiting sense. The following claims are intended to cover all genericand specific features described herein, as well as all statements of thescope of the present method and system, which, as a matter of language,might be said to fall therebetween.

1. An apparatus for extracting stem cells from fat tissue, the apparatuscomprising; a specimen container having a closed bottom and an open top;an electromechanical agitator inside the specimen container; an agitatorcap removably coupled and sealed to the electromechanical agitator andconfigured to seal the top of the specimen container to maintainspecimen sterility; and an aspiration cap removably coupled and sealedto the specimen container to maintain specimen sterility, the aspirationcap including a syringe with a fluid channel for specimen extractionfrom the specimen container.
 2. The apparatus of claim 1, wherein theaspiration cap comprises an air inlet configured to keep the pressureinside the tube at an atmospheric pressure such that specimen extractioncan be performed without generating undue negative pressure within thespecimen container.
 3. The apparatus of claim 1, wherein theelectromechanical agitator comprises a stator rod and a plurality ofrotors connected to the stator rod.
 4. The apparatus of claim 3, whereinthe edges of the plurality of rotors are round and smooth to minimizedamage to stem cells.
 5. The apparatus of claim 3, wherein theelectromechanical agitator further comprises a plurality of lateralstabilizing bars to provide counter spin stabilization of specimenwithin the specimen container or the specimen container comprises aplurality of fins on inner wall of the container and/or grooves on outerwalls of the container for improving efficiency of the electromechanicalagitator.
 6. The apparatus of claim 1, further comprising a sleevebearing embedded within the sealed agitator cap configured to reducefriction.
 7. The apparatus of claim 1, wherein the electromechanicalagitator comprises a rotary or linear ultrasonic/subsonic driver tocause rotary or linear agitation.
 8. A sealed biological retainingpacket containing acellular biological matrix with a cellular hydrationport through which the harvested stem cells according to claim 1 withinthe processed stromovascular fraction containing stem cells can beintroduced for adhesion/hydration to the matrix for subsequentreplantation.
 9. A method for sterilely extracting stem cells from fat,the method comprising: placing a specimen container containing fat in anultrasonic resonator; applying an external ultrasonic field to the fatto separate stem cells from the fat in a medical care room; placing thespecimen container in a centrifuge to concentrate the separated stemcells; removing the specimen container from the centrifuge; andextracting the concentrated stem cells from the specimen container,wherein the specimen retains sterility through the sequence of the abovesteps.
 10. The method of claim 9, wherein a total time for sterilelyextracting stem cells from the fat is less than 30 minutes.
 11. Themethod of claim 10, wherein the total time for sterilely extracting stemcells from the fat is less than 10 minutes.
 12. A method for extractingstem cells from fat, the method comprising: placing an agitator cap ontoan open end of a specimen container having an open end and closedbottom, the agitator cap being removably coupled to the specimencontainer and also coupled to a mechanical agitator; separating stemcells from fat in a specimen container by the mechanical agitator in anoperating sterile room; replacing the agitator cap with an aspirationcap onto the open end of the specimen container, the aspiration capbeing removably coupled to the specimen container and also coupled to asyringe configured for extracting samples from the specimen container;placing the specimen container with the aspiration cap in a centrifugeto concentrate the separated stem cells; removing the specimen containerfrom the centrifuge; and extracting the stem cells from the bottom ofthe specimen container, wherein the specimen retains sterility throughthe sequence of the above steps.
 13. The method of claim 12, wherein theaspiration cap comprises an air inlet for the specimen container.